This invention was made with support under Grant No. HL 47377-03 from the NIH. Accordingly, the United States Government has certain rights in the invention.
Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
The recent interest in using Buckminster fullerene (fullerene) derivatives in biological systems raises the possibility of their assay by immunological procedures. This, in turn, leads to the question of the ability of these unprecedented polygonal structures, made up solely of carbon atoms, to induce the production of specific antibodies. Immunization of mice with a C60 fullerene derivative conjugated to bovine thyroglobulin yielded a population of fullerene-specific antibodies of the IgG isotype, showing that the immune repertoire was diverse enough to recognize and process fullerenes as protein conjugates. The population of antibodies included a subpopulation that crossreacted with a C70 fullerene as determined by immune precipitation and ELISA procedures. These assays were made possible by the synthesis of water-soluble fullerene derivatives, including bovine and rabbit serum albumin conjugates and derivatives of trilysine and pentalysine, all of which were characterized as to the extent of substitution and their UV-Vis spectra. Possible interactions of fullerenes with the combining sites of IgG are discussed based on the physical chemistry of fullerenes and previously described protein-fullerene interactions. They remain to be confirmed by the isolation of mAbs for x-ray crystallographic studies.
Until 1985 there were only two known allotropic forms of carbon: graphite and diamond. In 1985, a novel allotrope was reported in which 60 carbon atoms were arranged as a truncated icosahedron, with 60 vertices and 32 faces, 12 of which were pentagonal and 20 hexagonal (1). It was dubbed Buckminsterfullerene (usually shortened to fullerene) because of its geodesic character, a name that has held through the present day. A detailed background of metallofullerenes is provided in section B.1 of the fourth series of experiments (infra).
Considerable activity followed this discovery particularly after procedures were developed to prepare fullerenes in workable quantities (2, 3). Various fullerene-based compounds have been prepared, and diverse uses were sought for them. Some were incorporated into photovoltaic cells (4) and nanotubes (5). Others were tested for biological activity (6), including antiviral (7, 8), antioxidant (9, 10), and chemotactic activities (11), and as neuroprotective agents in a mouse model of amyotrophic lateral sclerosis (12).
Practical application of fullerenes as biological or pharmacological agents requires that dosage and serum levels be capable of measurement, preferably by sensitive, simple immunological procedures. This, in turn, requires that specific antibodies to fullerenes be produced.
The clonal selection theory tells us that antigens elicit the production of antibodies by selecting for specific antibody producing cells already present in the repertoire of immunized animals (13). Although there is debate about the size of the xe2x80x9cavailablexe2x80x9d repertoire (14, 15), immunologists usually work on the assumption that the repertoire is diverse enough to be counted on to produce antibodies to xe2x80x9canyxe2x80x9d molecule a researcher may choose. This is, of course, an unreliable assumption, as experimental failures rarely find their way into the literature. The question that arises, therefore, is whether the immune repertoire is xe2x80x9ccompletexe2x80x9d enough (15) to recognize and respond to the unprecedented geodesic structure of the fullerenes or sufficient aspects of it-more particularly, whether the immune system can process a fullerene-protein conjugate and display the processed peptides for recognition by T cells to yield IgG antibodies. We report here that it does.
This invention provides an antibody which is specific for a fullerene or derivative thereof, wherein the fullerene is selected from the group consisting of a fullerene carbon compound having from 20 to 540 carbon atoms.
This invention provides an antibody which is specific for a single-walled fullerene nanotube.
This invention provides a monoclonal antibody which is specific for a fullerene or derivative thereof, wherein the fullerene carbon compound or derivative thereof comprises a C60 fullerene, said antibody comprising an amino acid heavy chain sequence (SEQ ID NO:2) and an amino acid light chain sequence (SEQ ID NO:4).
This invention provides an antibody which is specific for a multi-walled fullerene nanotube.
This invention provides nucleic acid molecules which encode the monoclonal antibodies which are specific for a fullerene or derivative thereof, wherein the fullerene is selected from the group consisting of a fullerene carbon compound having from 20 to 540 carbon atoms.
This invention provides a nucleic acid molecule which encodes the monoclonal antibody which is specific for a single-walled fullerene nanotube.
This invention provides a nucleic acid molecule which encodes the monoclonal antibody which is specific for a multi-walled fullerene nanotube.
This invention provides a hybridoma produced by the fusion of a mouse antibody-producing cell and a mouse myeloma, said hybridoma producing a monoclonal antibody which is specific for a fullerene.
This invention provides a hybridoma produced by the fusion of a mouse antibody-producing cell and a mouse myeloma which is designated 1-10F-8A and deposited with the American Type Culture Collection (ATCC) under Accession Number PTA-279, said hybridoma producing a monoclonal antibody which binds to fullerene C60.
This invention provides a mouse monoclonal antibody specific for a fullerene-C60 and produced by the mouse monoclonal antibody-producing hybridoma designated 1-10F-8A and deposited with the ATCC under Accession Number PTA-279.
This invention provides a mouse monoclonal antibody specific for a fullerene-C60 and produced by the mouse monoclonal anti-fullerene antibody-producing hybridoma designated 1-10F-8A and deposited with the ATCC under Accession Number PTA-279, said antibody comprising a heavy chain sequence (SEQ ID NO:2) and a light chain sequence (SEQ ID NO:4).
This invention provides an antibody which is specific for a fullerene or derivative thereof, wherein the fullerene is selected from the group consisting of a fullerene carbon compound having from 20 to 540 carbon atoms, wherein the antibody is a polyclonal antibody.
This invention provides a monoclonal antibody which binds to a single-walled fullerene nanotube.
This invention provides a monoclonal antibody which binds to a multi-walled fullerene nanotube.
This invention provides a polyclonal antibody which binds to a single-walled fullerene nanotube.
This invention provides a polyclonal antibody which binds to a multi-walled fullerene nanotube.
This invention provides an antibody specific for a fullerene, wherein the fullerene is selected from the group consisting of a fullerene carbon compound or derivative thereof comprising from 20 to 540 carbon atoms, wherein a radioactive material is encapsulated in the fullerene.
This invention provides a method of determining a serum concentration of fullerenes in a subject which comprises: a) determining an amount of antibody which binds to the fullerene in the absence of serum; b) incubating a serum sample from a subject with an antibody which binds to the fullerene to form an antibody-fullerene complex; c) determining the amount of antibody which binds to the fullerene in the presence of serum by detecting the amount of fullerene complex; d) comparing the amount determined in step (c) with the amount determined in step (a), thereby determining the serum concentration of the fullerene in the subject.
This invention provides a method of purifying a fullerene from a sample which comprises: a) preparing an affinity chromatography column to which are bound antibodies which bind to the fullerene; b) adding a sample to the affinity chromatography column so as to allow the sample to flow through the column, thereby permitting the fullerene to bind to the antibodies, thereby forming a fullerene-antibody complex on the column; and c) separating the fullerenes from the antibody-fullerene complex of step (b) by altering the pH, thereby purifying the fullerene from the sample.
This invention provides a method of preparing a nanoscale device which comprises manipulating a single-walled fullerene nanotube or nanotubes with the above-described antibody which is specific for a single-walled fullerene nanotube to assemble electronic or chemical components of the nanoscale device.
This invention provides a method of preparing a nanoscale device which comprises manipulating a multi-walled fullerene nanotube or nanotubes with the above-described antibody which is specific for a multi-walled fullerene nanotube to assemble electronic or chemical components of the nanoscale device.